Cell kinetics of mouse kidney using bromodeoxyuridine incorporation and flow cytometry: preparation and staining

The kidney is recognized as a dose-limiting tissue by certain radiation treatments. The relationship between the onset of compensatory proliferation in response to irradiation and the expression of functional damage is difficult to study because of the low cell turnover in slowly proliferating tissues. We report on a method to obtain a suitable cell preparation from mouse kidney for study by flow cytometry using the recently developed staining techniques for bromodeoxyuridine incorporation. The labelling index of 0.3% in untreated mouse kidney was easily measured because large numbers of cells could be analysed rapidly. We show that compensatory proliferation after unilateral nephrectomy remains elevated for up to 3 weeks after surgery. Using the BrdU/FCM technique we were able to measure the duration of the S phase in normal and nephrectomized kidneys which we found to be 8.5 hr in both cases. The estimates of potential doubling time were similar to the time scale observed to elapse before functional damage is observed in normal kidneys and those in which damage is precipitated by surgery.     

Separated parabiont reveals the fate and lifespan of peripheral-derived immune cells in normal and ischaemia-induced injured kidneys

Immune cell infiltration plays a key role in acute kidney injury (AKI) to chronic kidney disease (CKD) progression. T lymphocytes, neutrophils, monocytes/macrophages and other immune cells regulate inflammation, tissue remodelling and repair. To determine the kinetics of accumulation of various immune cell populations, we established an animal model combining parabiosis and separation surgery to explore the fate and lifespan of peripheral leucocytes that migrate to the kidney. We found that peripheral T lymphocytes could survive for a long time (more than 14 days), whereas peripheral neutrophils survived for a short time in both healthy and ischaemia-induced damaged kidneys. Nearly half of the peripheral-derived macrophages disappeared after 14 days in normal kidneys, while their existing time in the inflammatory kidneys was prolonged. A fraction of F4/80^high macrophages were renewed from the circulating monocyte pool. In addition, we found that after renal ischaemia reperfusion, neutrophils increased significantly in the early phase, and T lymphocytes mainly accumulated in the late stage, whereas macrophages infiltrated throughout AKI-CKD progression and were sustained longer in injured as opposed to normal kidneys. In conclusion, peripheral-derived macrophages, T lymphocytes and neutrophils exhibit different lifespans in the kidney, which may play different roles during AKI-CKD progression.     

Differential expression of secreted phosphoprotein 1 in response to estradiol-17β and in ovarian tumors in chickens

Roundabout 2 (Robo2) is a member of the membrane protein receptor family. The chemorepulsive effect of Slit2-Robo2 signaling plays vital roles in nervous system development and neuron migration. Slit2-Robo2 signaling is also important for maintaining the normal morphogenesis of the kidney and urinary collecting system, especially for the branching of the ureteric bud (UB) at the proper site. Slit2 or Robo2 mouse mutants exhibit multilobular kidneys, multiple ureters, and dilatation of the ureter, renal pelvis, and collecting duct system, which lead to vesicoureteral reflux. To understand the effect of Robo2 on kidney development, we used microinjection and electroporation to overexpress GFP-Robo2 in an in vitro embryonic kidney model. Our results show reduced UB branching and decreased glomerular number after in vitro Robo2 overexpression in the embryonic kidneys. We found fewer metanephric mesenchymal (MM) cells surrounding the UB but no abnormal morphology in the branching epithelial UB. Meanwhile, no significant change in MM proliferation or apoptosis was observed. These findings indicate that Robo2 is involved in the development of embryonic kidneys and that the normal expression of Robo2 can help maintain proper UB branching and glomerular morphogenesis. Overexpression of Robo2 leads to reduced UB branching caused by fewer surrounding MM cells, but MM cell apoptosis is not involved in this effect. Our study demonstrates that overexpression of Robo2 by microinjection in embryonic kidneys is an effective approach to study the function of Robo2.     

Ablation of Gadd45β ameliorates the inflammation and renal fibrosis caused by unilateral ureteral obstruction

The growth arrest and DNA damage‐inducible beta (Gadd45β) protein have been associated with various cellular functions, but its role in progressive renal disease is currently unknown. Here, we examined the effect of Gadd45β deletion on cell proliferation and apoptosis, inflammation, and renal fibrosis in an early chronic kidney disease (CKD) mouse model following unilateral ureteral obstruction (UUO). Wild‐type (WT) and Gadd45β‐knockout (KO) mice underwent either a sham operation or UUO and the kidneys were sampled eight days later. A histological assay revealed that ablation of Gadd45β ameliorated UUO‐induced renal injury. Cell proliferation was higher in Gadd45β KO mouse kidneys, but apoptosis was similar in both genotypes after UUO. Expression of pro‐inflammatory cytokines after UUO was down‐regulated in the kidneys from Gadd45β KO mice, whereas UUO‐mediated immune cell infiltration remained unchanged. The expression of pro‐inflammatory cytokines in response to LPS stimulation decreased in bone marrow‐derived macrophages from Gadd45β KO mice compared with that in WT mice. Importantly, UUO‐induced renal fibrosis was ameliorated in Gadd45β KO mice unlike in WT mice. Gadd45β was involved in TGF‐β signalling pathway regulation in kidney fibroblasts. Our findings demonstrate that Gadd45β plays a crucial role in renal injury and may be a therapeutic target for the treatment of CKD.     

PROLIFERATION KINETICS OF THE MOUSE BLADDER AFTER IRRADIATION

The proliferative response of the mouse bladder was investigated, using continuous labelling with tritiated thymidine, at various times after a single dose of radiation. Bladder epithelial and vascular endothelial cells were studied. The cell turnover rate in unirradiated epithelium and endothelium was found to be extremely slow (in excess of 1 year). Irradiation with a single dose of 25 Gy resulted in compensatory proliferation of the epithelium but the response was not initiated for many months. At 3 months after irradiation there was little difference from the control proliferation rate, but from 6 to 22 months after irradiation (the end of the study) there was a period of sustained rapid proliferation with the cell turnover time reduced to approximately 1 week. The increase in proliferative activity observed at 22 months was found to be dose—dependent. Endothelial cells in the blood vessels of the submucosa also showed an increased turnover rate after irradiation and the timing of this reponse was found to be similar to that of the epithelium. The onset of compensatory proliferation in both cell types was found to coincide with marked histological and functional changes in the bladder. In this slowly proliferating tissue, the onset of rapid compensatory proliferation after irradiation is delayed and occurs at the time that functional impairment is observed. This supports the postulate that proliferation is unlikely to contribute much to the sparing effect of prolonged fractionated radiotherapy in slowly dividing tissues.     

Membrane-bound ribosomes in kidney: methods of estimation and effect of compensatory renal growth

Membrane-bound ribosomes are thought to secrete protein for export and free ribosomes to secrete protein for intracellular use. The proportion of the total ribosomes that is bound to membranes in normal mouse kidneys has been estimated by three different methods, and the results have been compared with those obtained by a fourth method used by us previously. The most valid estimates appear to be those obtained (a) by comparison of radioactivity in peaks representing free and membrane-bound ribosomes on linear sucrose gradients after labeling for 24 hr with ¹⁴C-orotic acid, and (b) by measurements of optical density in free and bound ribosomes that had been separated by centrifugation on discontinuous gradients of 0.5 M/2.0 M sucrose. Analyses by these methods show that about 20–25% of the ribosomes in a postnuclear supernatant prepared from mouse kidneys, but only 10–15% of the ribosomes in a post-mitochondrial supernatant, are membrane-bound. About 75% of the bound ribosomes sediment as polysomes of many different sizes. The proportion of membrane-bound ribosomes and their aggregation into polysomes were unchanged in kidneys undergoing compensatory hypertrophy after removal of the opposite kidney. These experiments show that, unlike liver, kidney has a predominance of free ribosomes compared to bound ribosomes; those ribosomes that are membrane-bound do not become free during compensatory renal growth.     

Levels and patterns of 125I-labeled transferrin binding in mouse embryonic teeth and kidneys at various developmental stages

The iron-transporting serum glycoprotein, transferrin, is necessary for the cell proliferation, morphogenesis, and differentiation of mouse embryonic teeth and kidneys in organ culture. The stimulatory effect of transferrin is mediated by the binding of transferrin to its specific cell-surface receptor and by receptor-mediated endocytosis. Since, in both teeth and kidneys, the requirement for and responsiveness to transferrin depend on the developmental stage of the organ, we studied the binding of transferrin at various stages of tooth and kidney development by incubating tissues with 125I-labeled transferrin. The amount of bound transferrin was determined by measuring the tissue-incorporated radioactivity, and the binding sites were localized by autoradiography. During tooth development in vitro, the requirement for exogenous transferrin is lost as the teeth proceed from the early cap stage to the bell stage. The level of transferrin binding was found to decrease simultaneously, and in bell-stage teeth, the transferrin receptors were concentrated in the areas of most active cell proliferation. In kidneys, the number of transferrin receptors was highest at the stage during which the undifferentiated kidney mesenchyme becomes responsive to transferrin. These receptors were located in both the ureter epithelium and the metanephric mesenchyme, and they dramatically decreased in number with advancing kidney differentiation. The results of the present study indicate that, during the embryonic development of teeth and kidneys, the amount and localization of transferrin binding are correlated with cell proliferation. The number of transferrin receptors is highest during the developmental stages when cell proliferation is most active, and decreases with advancing differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of abnormal nuclei in renal proximal tubules after irradiation

The distribution and kinetics of proximal tubular cells with abnormally large nuclei, which were observed in irradiated mouse kidneys before any other obvious histological effects, were investigated. Six months after the administration of 13 or 15 Gy, little histopathological change was noted, in the kidneys of C3H mice; however, proliferation of proximal tubular cells was stimulated, and some of these cells had abnormally large nuclei. The relative DNA content of these large nuclei was measured with a quantitative image analysis system. Most of the large nuclear cells had more than diploid DNA content. The labeling index of the large nuclei was higher than that of unselected proximal tubular nuclei. These cells might be hyperploid cells that are dying after having gone through an abortive mitotic division. Examination and quantitation of these abnormal nuclei should be useful in elucidating the steps involved in cell loss in the proximal tubules after irradiation and as an assay for radiation damage to the kidney.     

Localization of monoclonal antibody TNT-1 in experimental kidney infarction of the mouse

An experimental kidney infarction model was developed in the mouse to study the uptake of a radiolabeled monoclonal antibody previously shown to bind to degenerating cells in malignant tumors. To determine if this approach is applicable to normal tissue and cell degeneration, kidney infarction was produced by clamping the mouse renal artery for 3 h using surgical procedures. Various groups of mice were injected with 131I-labeled TNT-1 F(ab')2 monoclonal antibody directed against nuclear histone antigens at varying intervals after surgery. Imaging, biodistribution, autoradiography, and histological studies were performed on each group of mice, including sham-operated controls, to quantitate the level of binding and localize the uptake of label in clamped and unclamped (contralateral) kidneys. As additional controls, clamped mice were administered radiolabeled irrelevant monoclonal antibody Lym-1 or mouse albumin. The results showed a marked selective uptake of radiolabeled TNT-1 F(ab')2 in the injured clamped kidney compared with the untreated kidney and other normal organs of the mouse. These studies define a model of normal organ necrosis that may be useful for study of the kinetics of antibody uptake in infarcted tissues.     

Kidney cell death in inflammation: The role of oxidative stress and mitochondria

Pyelonephritis is an infectious disease, and common treatment strategy is based on antibiotic therapy directed at the elimination of a pathogen. However, urinary tract infections are accompanied by inflammation and oxidative stress, which are major damaging factors, and therefore can serve as a target for therapeutic intervention. The goal of this study was to clarify the role of the mitochondrial reactive oxygen species (ROS) in kidney cell damage under experimental pyelonephritis. We investigated the mechanisms of inflammation and the role of mitochondria and oxidative stress in inflammation in kidney tissue using in vivo and in vitro models of pyelonephritis. We observed the development of oxidative stress in renal tubular epithelium in vitro, and resulting apoptotic cell death. This oxidative damage was caused by the leukocytes producing ROS after interaction with bacterial antigens. The essential role of mitochondria-mediated oxidative stress was confirmed using an experimental model of pyelonephritis in vivo. We revealed increased levels of malonic dialdehyde in kidneys of rats with experimental pyelonephritis that pointed to lipid peroxidation. Besides, high ROS levels were observed in blood leukocytes from rats with pyelonephritis. The mitochondria-targeted antioxidant SkQ1 significantly reduced the signs of kidney inflammatory injury, in particular the infiltration of neutrophils. Summarizing the data obtained, we assume the importance of mitochondrial ROS in different phases of acute pyelonephritis onset. Protection of kidney cells from infection-mediated damage can be attained by the induction of tolerance mechanisms and by antioxidant treatment.     

Increased glomerular Vwf after kidney irradiation is not due to increased biosynthesis or endothelial cell proliferation.

Kuin, A., Citarella, F., Oussoren, Y. G., Van der Wal, A. F., Dewit, L. G. H. and Stewart, F. A. Increased Glomerular Vwf after Kidney Irradiation is not due to Increased Biosynthesis or Endothelial Cell Proliferation. Radiat. Res. 156, 20-27 (2001).Irradiation of the kidney induces dose-dependent, progressive renal functional impairment, which is partly mediated by vascular damage. It has previously been demonstrated that reduced renal function is preceded by an increased amount of von Willebrand factor (Vwf) in the glomerulus. The underlying mechanism and significance of this observation are unknown but, since it is an important mediator of platelet adhesion, Vwf in increased amounts could be implicated in glomerular thrombosis, resulting in impairment of renal function. Increased Vwf could be the result of increased biosynthesis by endothelial cells, or from increased numbers of endothelial cells after compensatory proliferation induced by irradiation, or it could be secondary to other events. In the present study, expression levels of mRNA for glomerular Vwf and glomerular cell proliferation rates were measured in control mouse kidneys and after irradiation with a single dose of 16 Gy. There were no significant changes in mRNA ratios for Vwf/beta-actin at 10 to 30 weeks after irradiation compared with unirradiated samples, whereas increased amounts of Vwf protein were seen in the glomeruli at these times. Labeling studies with IdU or staining for Ki67 demonstrated that glomerular proliferation was increased from 10 to 30 weeks after irradiation. Despite the increased proliferation rates, there was an absence of glomerular hyperplasia and no increase in the endothelial cell surface coverage in the glomeruli. Staining with antibodies against smooth muscle actin (SMAalpha) revealed that the observed proliferation mainly involved mesangial cells. These results indicate that the increased presence of glomerular Vwf after irradiation is not due to an increased number of endothelial cells per glomerulus, or to an increased production of Vwf. It is presumably secondary to other events, such as increased release of Vwf by damaged endothelial cells or entrapment of Vwf in the irradiated mesangial matrix.     

Analysis of Kif5b Expression during Mouse Kidney Development

Recent studies showed that kidney-specific inactivation of Kif3a produces kidney cysts and renal failure, suggesting that kinesin-mediated intracellular transportation is important for the establishement and maintenance of renal epithelial cell polarity and normal nephron functions. Kif5b, one of the most conserved kinesin heavy chain, is the mouse homologue of the human ubiquitous Kinesin Heavy Chain (uKHC). In order to elucidate the role of Kif5b in kidney development and function, it is essential to establish its expression profile within the organ. Therefore, in this study, we examined the expression pattern of Kif5b in mouse kidney. Kidneys from embryonic (E) 12.5-, 16.5-dpc (days post coitus) mouse fetuses, from postnatal (P) day 0, 10, 20 pups and from adult mice were collected. The distribution of Kif5b was analyzed by immunostaining. The possible involvement of Kif5b in kidney development was investigated in conditional mutant mice by using a Cre-LoxP strategy. This study showed that the distribution of Kif5b displayed spatiotemporal changes during postnatal kidney development. In kidneys of new born mice, Kif5b was strongly expressed in all developing tubules and in the ureteric bud, but not in the glomerulus or in other early-developing structures, such as the cap mesenchyme, the comma-shaped body, and the S-shaped body. In kidneys of postnatal day 20 or of older mice, however, Kif5b was localized selectively in the basolateral domain of epithelial cells of the thick ascending loop of Henle, as well as of the distal convoluted tubule, with little expression being observed in the proximal tubule or in the collecting duct. Conditional knock-down of Kif5b in mouse kidney did not result in detectable morphological defects, but it did lead to a decrease in cell proliferation rate and also to a mislocalization of Na⁺/K⁺/-ATPase, indicating that although Kif5b is non-essential for kidney morphogenesis, it is important for nephron maturation.     

Renal Primordia Activate Kidney Regenerative Events in a Rat Model of Progressive Renal Disease

New intervention tools for severely damaged kidneys are in great demand to provide patients with a valid alternative to whole organ replacement. For repairing or replacing injured tissues, emerging approaches focus on using stem and progenitor cells. Embryonic kidneys represent an interesting option because, when transplanted to sites such as the renal capsule of healthy animals, they originate new renal structures. Here, we studied whether metanephroi possess developmental capacity when transplanted under the kidney capsule of MWF male rats, a model of spontaneous nephropathy. We found that six weeks post-transplantation, renal primordia developed glomeruli and tubuli able to filter blood and to produce urine in cyst-like structures. Newly developed metanephroi were able to initiate a regenerative-like process in host renal tissues adjacent to the graft in MWF male rats as indicated by an increase in cell proliferation and vascular density, accompanied by mRNA and protein upregulation of VEGF, FGF2, HGF, IGF-1 and Pax-2. The expression of SMP30 and NCAM was induced in tubular cells. Oxidative stress and apoptosis markedly decreased. Our study shows that embryonic kidneys generate functional nephrons when transplanted into animals with severe renal disease and at the same time activate events at least partly mimicking those observed in kidney tissues during renal regeneration.     

Autophagy plays a critical role in kidney tubule maintenance, aging and ischemia-reperfusion injury

Autophagy is responsible for the degradation of protein aggregates and damaged organelles. Several studies have reported increased autophagic activity in tubular cells after kidney injury. Here, we examine the role of tubular cell autophagy in vivo under both physiological conditions and stress using two different tubular-specific Atg5-knockout mouse models. While Atg5 deletion in distal tubule cells does not cause a significant alteration in kidney function, deleting Atg5 in both distal and proximal tubule cells results in impaired kidney function. Already under physiological conditions, Atg5-null tubule cells display a significant accumulation of p62 and oxidative stress markers. Strikingly, tubular cell Atg5-deficiency dramatically sensitizes the kidneys to ischemic injury, resulting in impaired kidney function, accumulation of damaged mitochondria as well as increased tubular cell apoptosis and proliferation, highlighting the critical role that autophagy plays in maintaining tubular cell integrity during stress conditions.     

Serum renotropic factor stimulates prostaglandin synthesis in primary cultures of rabbit kidney cells

Compensatory growth of the kidney occurs in response to a partial reduction in renal mass. This compensatory renal growth may be regulated by a circulating renotropic factor. Prostaglandin synthesis has been shown to be increased in kidneys undergoing compensatory renal growth in vivo. In the present study we observed that the addition of rabbit sera obtained after uninephrectomy enhanced DNA synthesis in primary cultures of rabbit kidney cells compared to sera obtained prenephrectomy. The stimulated kidney cells produced more prostaglandin E2 than control cells. Furthermore, the addition of prostaglandin E2 to rabbit kidney cells in the presence of control sera also stimulated DNA synthesis. These results provide further evidence that prostaglandins may participate in the biological events which regulate renal growth in response to a circulating renotropic factor.     

Global gene expression profiling in early-stage polycystic kidney disease in the Han:SPRD Cy rat identifies a role for RXR signaling

Han:SPRD Cy is a spontaneous rat model of polycystic kidney disease (PKD) caused by a missense mutation in Pkdr1. Cystogenesis in this model is not clearly understood. In the current study, we performed global gene expression profiling in early-stage PKD cyst development in Cy/Cy kidneys and normal (+/+) kidneys at 3 and 7 days of postnatal age. Expression profiles were determined by microarray analysis, followed by validation with real-time RT-PCR. Genes were selected with over 1.5-fold expression changes compared with age-matched +/+ kidneys for canonical pathway analysis. We found nine pathways in common between 3- and 7-day Cy/Cy kidneys. Three significantly changed pathways were designated "Vitamin D Receptor (VDR)/Retinoid X Receptor (RXR) Activation," "LPS/IL-1-Mediated Inhibition of RXR Function," and "Liver X Receptor (LXR)/RXR Activation." These results suggest that RXR-mediated signaling is significantly altered in developing kidneys with mutated Pkdr1. In gene ontology analysis, the functions of these RXR-related genes were found to be involved in regulating cell proliferation and organ morphogenesis. With real-time RT-PCR analysis, the upregulation of Ptx2, Alox15b, OSP, and PCNA, major markers of cell proliferation associated with the RXR pathway, were confirmed in 3- and 7-day Cy/Cy kidneys compared with 3-day +/+ kidneys. The increased RXR protein was observed in both the nucleus and cytoplasm of cystic epithelial cells in early-stage Cy/Cy kidneys, and the RXR-positive cells were strongly positive for PCNA staining. Taken together, cell proliferation and organ morphogenesis signals transduced by RXR-mediated pathways may have important roles for cystogenesis in early-stage PKD in this Pkdr1-mutated Cy rat.     

Polycystin-1 and polycystin-2 regulate the cell cycle through the helix-loop-helix inhibitor Id2

Autosomal-dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and is characterized by progressive cyst formation and ultimate loss of renal function. Increased cell proliferation is a key feature of the disease. Here, we show that the ADPKD protein polycystin-2 (PC2) regulates the cell cycle through direct interaction with Id2, a member of the helix-loop-helix (HLH) protein family that is known to regulate cell proliferation and differentiation. Id2 expression suppresses the induction of a cyclin-dependent kinase inhibitor, p21, by either polycystin-1 (PC1) or PC2. The PC2-Id2 interaction is regulated by PC1-dependent phosphorylation of PC2. Enhanced Id2 nuclear localization is seen in human and mouse cystic kidneys. Inhibition of Id2 expression by RNA interference corrects the hyperproliferative phenotype of PC1 mutant cells. We propose that Id2 has a crucial role in cell-cycle regulation that is mediated by PC1 and PC2.     

Reirradiation at long time intervals in mouse kidney: a comparison between experimental results and the predictions of the F-type tissue model

The tolerance of a late-responding tissue to reirradiation after long time intervals has been analysed using the F-type tissue model. In this model the tissue is composed of identical cells, each of which is capable of extensive proliferation and of tissue-specific function. The model was adapted to calculate the response to two fractions of radiation given in a variable overall time. For two equal doses of radiation the repair of tissue damage after the first fraction could be detected theoretically by a change in the rate of cell depletion after retreatment and by an increase in the minimum cell number attained. For an 'experimental set-up', in which a constant first dose was followed by a range of retreatment doses in a variable overall time, the repair of tissue damage theoretically could be detected most sensitively by a shift of the dose-response curves to higher retreatment doses as the time interval between the two doses was increased. A prerequisite for a proper comparison of these dose-response curves was that the responses were evaluated at times after the first dose determined by the minimal latency times after high retreatment doses. From a comparison of these theoretical results with experimental findings for mouse kidneys it was concluded that no recovery of tissue function took place over a 6-month period. Instead it appeared that the kidneys had become more sensitive to irradiation over this period.